Abstract A006: Direct immunosuppressive effect of the matricellular protein fibulin-3 on tumor-associated macrophages in glioblastoma

Abstract Background: The growth of primary malignant brain tumors (glioblastoma, GBM) leads to extensive changes in the composition and physical properties of the neural extracellular matrix (ECM). It is hypothesized that these changes modify the behavior of tumor-associated microglia and infiltrated macrophages (TAMs), which are the predominant immune cell population in GBM. Fibulin-3 is an ECM glycoprotein uniquely enriched in GBM, compared to the normal brain, that promotes tumor growth, vascularization and survival of the GBM stem cell (GSC) population. We have recently identified an autocrine effect of GSC-secreted fibulin-3 that dampens anti-tumor immune responses of TAMs against tumor cells. Here we describe a novel, complementary, immunosuppressive effect of this ECM protein by direct action on macrophages. Methods: The effects of fibulin-3 on macrophage cell viability, proliferation and migration, were tested with conventional assays using the myeloid cell lines Raw264. 7, THP-1, and HMC3 (microglia). Human peripheral blood mononuclear cells and mouse bone marrow derived macrophages were isolated and treated with purified fibulin-3 to analyze signaling pathways and differential gene expression using cytokine arrays, phospho-RTK array, Western blotting and RNA sequencing. Tumor cells were co-cultured with fibulin-3 (or vehicle) -treated macrophages to assess their phagocytic activity. In vitro macrophage polarization was induced with cytokine cocktails to assess fibulin-3 effects. Genetic knockdown and pharmacological inhibition of the collagen receptor DDR2 were performed to test the requirement of this receptor for fibulin-3 effects in myeloid cells. Results: Fibulin-3 did not affect the proliferation of macrophage or microglia but increased the transmigration of these cells and decreased their phagocytic activity against tumor cells. These phenotypic changes induced by fibulin-3 were accompanied by increased expression of pro-migratory cytokines, upregulation of the phagocytosis inhibitor SIRP1α, and decreased expression of T cell co-stimulatory signals (CD137L, CD70). Fibulin-3 did not activate Notch/NF-kB signaling in macrophages as previously reported in glioma cells; instead, fibulin-3 increased the phosphorylation of the immunosuppressive receptor DDR2, as well as signaling mediated by Src/MAPK/RUNX2 activation. Fibulin-3 dampened the responses of cultured macrophages to an MIFNg) -polarizing cytokine cocktail; this effect was partially dependent on DDR2 expression. Accordingly, the upregulation of SIRP1a by fibulin-3 was abolished by genetic or pharmacologic inhibition of DDR2. RNAseq data suggested a phenotypic change induced by fibulin-3 in macrophages corresponding to acquisition of ECM-remodeling, pro-angiogenic, and pro-fibrotic mechanisms. Conclusions: Our findings suggest that fibulin-3 secreted by tumor cells has direct immunosuppressive functions on the TAM population present in GBM, making this protein an attractive target to boost immune responses against the tumor. Citation Format: Soham Mitra, Somanath Kundu, Abigail Venskus, Mariano S. Viapiano. Direct immunosuppressive effect of the matricellular protein fibulin-3 on tumor-associated macrophages in glioblastoma [abstract. In: Proceedings of the AACR Special Conference in Cancer Research: Brain Cancer; 2026 Mar 23-25; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2026;86 (6Suppl): Abstract nr A006.